GC Analysis of Glycols in Toothpaste Using Agilent J&W DB-Wax Ultra Inert Capillary GC Columns

Application Note

Flavors and Fragrances, Consumer Products

Author Abstract

Yun Zou This application note demonstrates the excellent inertness performance of Agilent Technologies, Ltd an Agilent J&W DB-Wax Ultra Inert GC column in the analysis of glycols in Shanghai toothpaste. DB-Wax UI columns provide better column durability and more consistent analytical results compared with other PEG stationary phase GC columns.

Introduction

Typical toothpaste formulas are composed of various components including abrasives, fluoride, detergents, and a list of US-FDA approved flavors and sweeteners. Common moisturizers and sweeteners in toothpaste are propylene glycol, sorbitol, and glycerin.

Diethylene glycol (DEG) is an organic solvent often used in antifreeze [1,2], and unfortunately is chemically similar to propylene glycol and glycerin. DEG is classified as a toxic material, and has been indicated as a contributor in multiple systemic disorders leading to acute kidney failure and death [3]. DEG has physical and chemical properties close to the properties of glycerin and propylene glycol. Commercially, both glycerin and propylene glycol are more expensive than DEG. This facilitates the illegal use of DEG as a counterfeit substitute for glycerin in some nations, where it is sold as a component in cough syrup and toothpaste.

DEG is synthesized from the reaction of ethylene oxide with ethylene glycol, therefore, DEG can contain ethylene glycol (EG), a toxic impurity [4]. The US-FDA is concerned about potential risks from chronic exposure to DEG. There is also a concern, in certain populations, of DEG exposure to children and individuals with kidney or liver disease. DEG in toothpaste has a low but meaningful risk of toxicity and injury. The US-FDA has issued a recommended method to determine the levels of DEG and EG in toothpastes [5]. Low levels of DEG, EG, 1,2-propanediol, 1, 3-propanediol, and Performance test sample glycerin were tested in this method. Due to the chemical Low standard, 0.1 mg/mL of each analyte including properties of these compounds, particularly the multiple 1,2-propanediol, 1,3-propanediol (internal standard), DEG, EG, active hydroxyl (-OH) functional groups, their peaks often and glycerin was prepared in 50% (v/v) aqueous acetonitrile. exhibit tailing when analyzed using traditional polyethylene glycol (PEG) stationary phase GC columns. Inertness of the Sample preparation column is important to achieve consistent and reliable results. Agilent J&W DB-Wax Ultra Inert columns are engineered for Matrix blank better peak shapes, and are rigorously tested with demanding Approximately 1.0 g of toothpaste was weighed into a 15 mL probes to verify best-in-class inertness. This application note polypropylene centrifuge tube. demonstrates the use of J&W DB-Wax Ultra Inert columns in the analysis of the active compounds in toothpaste with Five milliliters of water was added, then vortexed for 1 minute. GC/MSD. It also compares the performance with PEG Foam started to appear, and 2 × 2.5 mL of acetonitrile columns from other suppliers. was added to suppress the foam. The sample was mixed thoroughly, and centrifuged at 4,000 rpm for 10 minutes. Then, Experimental 0.50 mL of the supernatant was transferred to an autosampler vial, to which 0.05 mL of the internal standard was added. The sample preparation was carried out according to the Spiked sample US FDA method [5]. The toothpaste sample was obtained from a local supermarket. Approximately 1.0 g of toothpaste was weighed into a 15 mL polypropylene centrifuge tube. Then, 0.20 mL of mixed Chemicals and reagents standard was added. The rest of the procedure was followed as previously described. All reagents and solvents were HPLC or analytical grade. Acetonitrile (ACN) was from J&K Scientific (Beijing, China). Water was from J. T. Baker. Chemicals including Instrumentation 1,2-propanediol, 1,3-propanediol, ethylene glycol (EG), Table 1 shows the instruments and conditions, and Table 2 diethylene glycol (DEG), and glycerin were from ANPEL lists the consumable supplies flow path. Scientific Instrument Co. Ltd (Shanghai, China). Table 1. Conditions. Solutions and standards Parameter Value Internal standard (IS) GC system Agilent 7890B GC with an Agilent 5977A Series GC/MSD A 1, 3-propanediol solution was prepared at 5.0 mg/mL in System Column Agilent J&W DB-Wax UI, 30 m × 0.25 mm, 0.25 µm 50% (v/v) aqueous acetonitrile. (p/n 122–7032UI) Wax columns from other suppliers, Mixed standard 30 m × 0.25 mm, 0.25 µm Tubing Agilent Ultimate Plus deactivated FS tubing, DEG and EG were prepared in 50% (v/v) aqueous acetonitrile 5 m × 0.25 mm (p/n CP802505) to give 5.0 mg/mL of each component. Autosampler Agilent 7683B autosampler and sample tray, 5 µL syringe (p/n G4513-80213), 1 µL injection volume Low standards Carrier gas Helium, 35 cm/s constant flow mode The mixed standard was diluted to 0.1 mg/mL of each Inlet Split/splitless, 250 °C, split ratio 20:1 analyte, and 0.50 mL of this standard was transferred to Oven 100 °C (1 min) to 250 °C at 10 °C/min (hold 4.00 min) the autosampler vial, followed by the addition of 0.05 mL of MSD Agilent 5977A Series GC/MSD System internal standard. Solvent delay 4.0 min MS temp 230 °C (source), 150 °C (quad) High standards Transfer Line 250 °C The mixed standard was diluted to 0.5 mg/mL of each MS EI, Scan mode, Scan 29–400 amu analyte, and 0.50 mL of this standard was transferred to the autosampler vial, followed by the addition of 0.05 mL of internal standard.

2 Table 2. Flow path supplies. commonly used as industrial solvents and in antifreeze solutions. DEG and EG should not be found in toothpaste Parameter Value formulations. 1,2-propanediol is a related compound, and Vials Amber, write-on spot, certified, 2 mL, screw top vial packs (p/n 5182–0554) glycerin is a common component found in toothpaste. Septa Nonstick BTO septa (p/n 5183–4757) 1,3-propanediol is used as an internal standard (IS) according to the US-FDA method. Due to the multiple active hydroxyl Column nut Self-tightening, inlet/detector (p/n 5190–6194) Self-tightening, for MS interface (p/n 5190–5233) (-OH) functional groups in these compounds, traditionally Internal nut CFT capillary fitting (p/n G2855-20530) it has been quite challenging to achieve good peak shape. Union Ultimate union (p/n G3182-60581) Figure 1 shows the symmetrical peak shapes for these active Ferrules 15% graphite: 85% Vespel, short, 0.4 mm id, for compounds at 0.1 mg/mL. This symmetry indicates the high 0.1 to 0.25 mm columns (10/pk, p/n 5181–3323) level of inertness of the J&W DB-Wax Ultra Inert GC column. UltiMetal Plus Flexible Metal, for 0.1 to 0.25 mm columns (10/pk, p/n G3188-27501) The US-FDA method uses a 30 m × 0.25 mm, 0.25 µm Wax Liner Agilent Ultra Inert split liner with glass wool (p/n 5190–2295) column with a 5 m guard column to evaluate the presence of Inlet seal Ultra Inert, gold-plated, with washer (p/n 5190–6144) DEG and EG at 1 mg/g (0.1 % by weight) and above. A 5 m × 0.25 mm Agilent Ultimate Plus deactivated FS tubing was used as a guard column connected with an Results and Discussion Agilent DB‑Wax Ultra Inert 30 m × 0.25 mm, 0.25 µm GC column in this application. Low (0.1 mg/mL) and high The purpose of these tests was to evaluate the inertness standards (0.5 mg/mL) were analyzed using GC/MSD in full performance of Agilent J&W DB-Wax Ultra Inert columns scan mode with an Agilent J&W DB-Wax UI GC column and relative to Wax columns from other suppliers, using a typical a DB-Wax UI GC column attached to a 5 m guard column. The toothpaste analysis. DB-Wax UI GC column with guard column delivered similar symmetrical peak shape and response of EG and DEG as a Toothpaste analysis single DB-Wax UI GC column when analyzing low and high standards. This is a result of the advanced Agilent inert flow Glycerin and propylene glycol (PG) are widely used in path technology. 1,3-propanediol is a challenging compound, toothpaste. Glycerin, PG, DEG, and EG have similar physical but the peak exhibited relatively little tailing on a DB-Wax UI properties, such as sweetness and viscosity, which facilitates GC column with the guard column in-line. the adulteration process. DEG and EG are target compounds for this analysis, because they are known poisons that are

×105 40

Diethylene glycol 20 1,2-Propanediol 15 1,3-Propanediol (IS) Glycerin Ethylene glycol 10

6 78910 11 12 13 Time (min) Figure 1. GC/MS total ion chromatogram of analytes and related compounds at a concentration of 0.1 mg/mL. Performed on an Agilent J&W DB-Wax Ultra Inert, 30 m × 0.25 mm, 0.25 μm GC column.

3 Figure 2 compares the results observed with and without the guard column installed on a DB-Wax Ultra Inert 30 m × 0.25 mm, 0.25 µm GC column.

Agilent DB-Wax UI column + Agilent Ultimate Plus deactivated FS tubing Agilent DB-Wax UI column + Agilent Ultimate Plus deactivated FS tubing ×105 ×105 50 A B DEG 1,3-Propanediol (IS) 80 Low standards High standards 40 60 1,3-Propanediol (IS) 30 EG 40 20 DEG EG 10 20

0 0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 Time (min) Time (min)

Agilent DB-Wax UI column Agilent DB-Wax UI column ×105 1,3-Propanediol (IS) ×105 50 C D DEG 80 Low standards High standards 40 60 1,3-Propanediol (IS) 30 EG 40 20 DEG EG 10 20

0 0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 10.0 10.5 11.0 11.5 Time (min) Time (min) Figure 2. GC/MS total ion chromatograms of low (0.1 mg/mL) and high standards (0.5 mg/mL), on an Agilent DB‑Wax Ultra Inert GC column attached to a guard column, and an Agilent DB-WAX Ultra Inert GC column not attached to a guard column.

4 A 1 mg/g amount of DEG and EG spiked into a toothpaste sample were analyzed using a DB-Wax UI GC column with a guard column. Figure 3 shows the chromatograms of matrix blank and spiked sample. No DEG and EG was present in the toothpaste matrix blank sample. Although many components including menthyl acetate, 1,2-propanediol, menthol, glycerin, thymol, and methylparaben were identified in the samples, they did not interfere with analysis of DEG and EG. Excellent peak shape was observed for all these active compounds in Figure 3.

Peak ID 1. Menthyl acetate ×105 Spiked sample 2. 1,2-Propanediol 160 3. Ethylene glycol 2 4 140 4. Menthol 120 100 5. 1,3-Propanediol 80 (DEG) 6. Diethylene glycol 60 EG (IS) 6 9 7. Glycerin 1 3 40 5 8 8. Thymol 20 7 0 9. Methylparaben 6810 12 14 16 18 20 Time (min) ×105 Toothpaste blank 160 2 4 140 120 100 80 60 (IS) 9 40 1 5 8 20 7 0 6810 12 14 16 18 20 Time (min) Figure 3. GC/MS total ion chromatogram of spike 1.0 mg/g of EG and DEG in toothpaste sample and toothpaste matrix blank

Comparison with other Wax columns According to the US-FDA method, the final oven temperature of this application is 250 °C. Inertness performance of Wax All columns were tested using a performance test sample. columns from different suppliers deteriorated differently after This consisted of 0.1 mg/mL of each analyte including conditioning the column for more than 2 hours to get an 1,2-propanediol, 1,3-propanediol, DEG, EG, and glycerin acceptable baseline. prepared in 50% (v/v) aqueous acetonitrile. The system was inspected, and carefully cleaned, if necessary, before each Figures 4 and 5 show comparison results. In the first few test. For consistency, new gold seals and liners were used for injections, wax columns from Supplier A and Supplier B initially each test. showed essentially similar performance as DB‑Wax UI. After 10 injections, there was noticeable tailing and reduced Normally, the first analysis on each Wax column showed an response of 1,3-propanediol in the chromatograms for acceptable or good peak shape. After a few injections, the suppliers A and B. peak shapes of EG, DEG, and 1,2-propanediol changed little. However, the peak shapes of 1,3-propanediol (peak 3) and High inertness and the improved thermal longevity of the glycerin (peak 5) deteriorated to different degrees due to the DB-Wax UI provided better peak shape and more durable variable inertness performance and thermal stability of each analytical results than was achievable on the other Wax Wax column. columns evaluated.

5 ×105 1,2-Propanediol 3rd injection 160 Agilent DB-WAX UI 140 1,3-Propanediol 120 Glycerin EG 100 80 10th injection 60 40 20 678910 11 12 13 Time (min)

×105 1,2-Propanediol DEG 160 3rd injection Supplier A WAX column 140 EG 120 1,3-Propanediol Glycerin 100 10th injection 80 60 40 20 678910 11 12 13 Time (min)

Figure 4. Chromatograms of performance test mixture on an Agilent DB-WAX UI and a Wax column from Supplier “A”.

Peak ID 1. 1,2-Propanediol 2. EG 3. 1,3-Propanediol 5 5 ×10 4. DEG ×10 4 22 Agilent DB-WAX UI 4 Agilent DB-WAX UI 5. Glycerin 22 18 1 1 5th injection 18 15th injection 14 3 2 14 2 10 5 3 10 6 5 6 2 2 0 6 78910 11 12 13 0 678910 11 12 13 Time (min) ×105 ×105 Supplier B WAX-MS Supplier B WAX-MS 4 22 4 22 1 1 18 18 15th injection 3 5th injection 14 2 14 2 10 10 3 5 6 6 5 2 2 0 0 6 78910 11 12 13 678910 11 12 13 Time (min) Time (min) Figure 5. Chromatograms of performance test mixture on an Agilent DB-WAX UI and a Wax column from Supplier B.

6 Conclusions References

Inertness performance of Agilent J&W DB-Wax Ultra inert 1. Marraffa, J. M.; Holland, M. G.; Stork, C. M.; Hoy, C. D.; GC column was evaluated by analyzing glycols in toothpaste Hodgman, M. J. Diethylene Glycol: Widely Used Solvent samples using GC/MSD. Presents Serious Poisoning Potential. J. of Emergency Med. 2007, 06, No. 025. Compared with other vendors’ Wax columns, J&W DB-Wax Ultra inert GC columns delivered better peak shape, and more 2. Reynolds, J. E. F.; Parfitt, K.; Parsons, A. V.; durable and consistent analytical results. Combining all the Sweetman, S. C. Martindale, The Complete Drugs inert flow path components, including Agilent Ultimate Plus Reference. Thirty–Fifth Edition: 2007, 1754–1756, 2100, Deactivated FS tubing as a guard column, it ensures excellent 2152, 2118, 2088. performance and reliable results for active compound 3. Kraut, J. A.; Kurtz, I. Toxic Alcohol Ingestions: Clinical analysis. Features, Diagnosis, and Management. J. Am. Soc. of Nephrology 2008, 3, 208–225. 4. Dr. Temple, W. A. DEG – draft poisons information monograph for peer review. International Programme on Chemical Safety, October 2007. 5. FDA method, Gas Chromatography-GC-MS Screening Procedure for the Presence of Diethylene Glycol and Ethylene Glycol in Toothpaste. http://www.fda.gov/Food/ ScienceResearch/ For More Information

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